Construction Of Recombinant Eukaryotic Expression Vector Of MMP-2 And Its Role In SMMC-7721 Hepatocellular Carcinoma Cells

Objective: To construct a recombinant eukaryotic expression vector of human MMP-2 and observe the effect of its expression protein on the migration and invasion of hepatocellular carcinoma cells,and to explore the above-mentioned mechanism.Methods: The liver cancer SMMC-7721 cells were used as the research model.The recombinant eukaryotic expression vector pEYFP-MMP-2 was constructed by gene recombination technology.The pEYFP-MMP-2 transfected model cells expressing MMP-2 fusion protein(MMP-2-YFP).RNAi inhibits endogenous MMP-2 expression in model cells.The experiment was divided into blank control group(Control),MMP-2 silencing control group(siCON),MMP-2 silencing group(siMMP-2),MMP-2 fusion protein control group(ovNC),MMP-2 fusion protein group(ovMMP-2).The scratch test detects cell migration ability.The invasive chamber assay to detect cell invasion ability.The calpain-specific inhibitor calpeptin inhibits calpain activity.The protein imprinting assay detects the expression changes of MMP-2,MMP-2 fusion protein,E-cadherin,N-cadherin and Vimentin proteins.Results(1)The MMP-2 eukaryotic expression vector pEYFP-MMP-2 was constructed and the MMP-2 fusion protein was highly expressed in model cells.(2)MMP-2 siRNA significantly inhibited the endogenous expression of MMP-2 in hepatocellular carcinoma cells(p<0.01).(3)Compared with siCON group,the mobility of siMMP-2 group was significantly decreased at 12 h,24h and 48 h,and the invasion rate was significantly decreased at 48h(p<0.01).Compared with ovNC group,the mobility of ovMMP-2 group was significantly increased at 12 h,24h and 48 h,and the invasion rate was significantly increased at 48h(p<0.05).(4)Compared with the siCON group,the expression of E-cadherin in siMMP-2 group was significantly up-regulated,and the expression of N-cadherin and Vimentin was down-regulated(p<0.01).Compared with the ovNC group,the expression of E-cadherin was significantly down-regulated,and the expression of N-cadherin and Vimentin was significantly up-regulated in the ovMMP-2 group(p<0.01).(5)Calpeptin pretreatment significantly reduced cell migration and invasion rate in ovMMP-2 group(p<0.01).(6)Calpeptin pretreatment significantly up-regulated the expression of E-cadherin,and down-regulated the expression of N-cadherin and Vimentin in ovMMP-2 group(p<0.01).Conclusion MMP-2 eukaryotic expression vector pEYFP-MMP-2 was successfully constructed,transfected into hepatocellular carcinoma cells highly expressed MMP-2 fusion protein.MMP-2 fusion protein can mediate migration,invasion and EMT of hepatocellular carcinoma cells by Calpain.